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Protein kinase D1 (PKD1), a ubiquitously expressed serine/threonine kinase, regulates diverse cellular processes such as oxidative stress, gene expression, cell survival, vesicle trafficking, Ag receptor signaling, and pattern recognition receptor signaling. We found previously that exposure to hypersensitivity pneumonitis (HP) inciting Ag Saccharopolyspora rectivirgula leads to the activation of PKD1 in a MyD88-dependent manner in various types of murine cells in vitro and in the mouse lung in vivo. However, it is currently unknown whether PKD1 plays a role in the S. rectivirgula–induced HP. In this study, we investigated contributions of PKD1 on the S. rectivirgula–induced HP using conditional PKD1-insufficient mice. Compared to control PKD1-sufficient mice, PKD1-insufficient mice showed substantially suppressed activation of MAPKs and NF-κB, expression of cytokines and chemokines, and neutrophilic alveolitis after single intranasal exposure to S. rectivirgula. The significantly reduced levels of alveolitis, MHC class II surface expression on neutrophils and macrophages, and IL-17A and CXCL9 expression in lung tissue were observed in the PKD1-insufficient mice repeatedly exposed to S. rectivirgula for 5 wk. PKD1-insuficient mice exposed to S. rectivirgula for 5 wk also showed reduced granuloma formation. Our results demonstrate that PKD1 plays an essential role in the initial proinflammatory responses and neutrophil influx in the lung after exposure to S. rectivirgula and substantially contribute to the development of HP caused by repeated exposure to S. rectivirgula. Our findings suggest that PKD1 can be an attractive new molecular target for therapy of S. rectivirgula–induced HP.

Long noncoding RNAs (lncRNAs) are a class of noncoding RNA, involved in regulation of diverse physiological and pathological processes. Ovarian cancer is the leading cause of death among all gynecological malignancies in the world and its underlying mechanism is still unclear. LncRNAs exhibit multiple biological functions in various stages of ovarian cancer development. We will discuss and summarize the new and important lncRNAs and their involvement in disease, which might represent promising therapeutic targets. Therapeutic intervention based on silencing or functional inhibition of target lncRNAs will be beneficial for ovarian cancer patients.

Background: Colorectal carcinoma (CRC) is the second leading cause of cancer related deaths in the United States. The five-year survival rate of patients diagnosed with distant stages declines to 14%, which is of concern when compared to 90% for localized-stage and 71% for regional stage disease. This necessitates the need for early diagnostic biomarkers, to minimize poor drug response/resistance, recurrence, metastasis and mortality. Disproportionate biochemical stressors increase the risk of developing CRC and its progression by influencing molecular drivers within the coding and noncoding parts of the genome. Thus, understanding the biological mechanism of these stress factors on the molecular drivers of this disease can provide pivotal information pertinent to CRC development and progression. Recently, our laboratory has identified a novel long noncoding RNA (lncRNA) namely, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), which is highly over-expressed in CRC and is involved in its pathogenesis and is regulated by transcription factor Nuclear Factor of Activated T cell 1 (NFATc1). Methods: Archived human CRC tissues were stained using a recently standardized Z-probe technology. TCGA database of ~600 CRC patients was also analyzed using the bioinformatic approach. CRC cell lines were profiled for MALAT1 expression using RT-PCR. Lentiviral constructs were used to generate stable lncRNA MALAT1 expressing cell lines. CRISPR/Cas9 constructs were used to knockdown lncRNA MALAT1 and NFATc1. Mouse model was used to verify the stress induced expression of MALAT1 and NFATc1. ReCLP (Reversible Cross-Linked Precipitation) and iRAP (invitro RNA Antisense Proteomics) studies are in progress to identify the associated proteins and complexes. Results: RNAScope analysis showed MALAT1 to be highly over-expressed in human CRC tissues. MALAT1 expression increased with stage and negatively correlated with the tumor size. TCGA database analysis confirmed our findings. Multiple NFATc1 binding site were identified by ChIPseq database. Overexpression of transcription factor NFATc1 upregulated lncRNA MALAT1 expression. CRISPR/Cas9 based knockdown of NFATc1 downregulated NFATC1 and lncRNA MALAT1, but vice versa was not true, indicating NFATc1 to be upstream of lncRNA MALAT1. Also, expression of MALAT1, NFATc1 and IL-6 were highly upregulated by biochemical stressor cortisol in mouse colonic tissues. Further studies are in progress for direct association of NFATc1 on MALAT1 promoter and mechanism by which the stress factor regulate NFATc1 expression and hence lncRNA MALAT1 expression. Conclusion: This study helps to understand influence of biochemical stress factors on long noncoding RNA MALAT1 and transcription factor NFATc1 etiology. Early diagnosis of these molecular markers will help in designing novel preventive/therapeutic strategies to reduce CRC progression, metastasis and hence mortality. Citation Format: Kyle Doxtater, Chidi Zacheaus, Radhika Sekhri, Utkarsh K. Mishra, Zachary E. Stiles, Nitish Mishra, Chittibabu Guda, Nadeem Zafar, Mahul Amin, Pradeep Shukla, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan, Manish K. Tripathi. Stress regulated role of lncRNA Malat1 in colorectal cancer progression and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3142.

Formalin-fixed paraffin embedded (FFPE) tissues are a valuable resource for biomarker discovery in order to understand the etiology of different cancers and many other diseases. Proteins are the biomarkers of interest with respect to FFPE tissues as RNA degradation is the major challenge in these tissue samples. Recently, non-protein coding transcripts, long non-coding RNAs (lncRNAs), have gained significant attention due to their important biological actions and potential involvement in cancer. RNA sequencing (RNA-seq) or quantitative reverse transcription-polymerase chain reaction (qRT-PCR) are the only validated methods to evaluate and study lncRNA expression and neither of them provides visual representation as immunohistochemistry (IHC) provides for proteins. We have standardized and are reporting a sensitive Z probe based in situ hybridization method to visually identify and quantify lncRNA in FFPE tissues. This assay is highly sensitive and identifies transcripts visible within different cell types and tumors. We have detected a scarcely expressed tumor suppressor lncRNA NRON (non-coding repressor of nuclear factor of activated T-cells (NFAT)), a moderately expressed oncogenic lncRNA UCA1 (urothelial cancer associated 1), and a highly studied and expressed lncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) in different cancers. High MALAT1 staining was found in colorectal, breast and pancreatic cancer. Additionally, we have observed an increase in MALAT1 expression in different stages of colorectal cancer.

Colorectal Cancer(CRC) is one of the leading cause of cancer related death in the United States today in both men and women. With an expected 50,260 deaths in 2017 it is the second leading cause of cancer related deaths among men and the third among women in the United states. Often in colorectal cancer it is not the primary tumor that is the killer, but it is the metastatic sites that develop in the later stages of the disease that is the true killer. About 90% of all cancer related deaths are due to the development of metastatic sites in the body. What is metastasis? Metastasis is the transfer of disease from the primary tumor site to another unconnected site within the body through a process called the metastatic cascade. Metastasis develops slowly over time, due to it being an inefficient process having to overcome several natural defenses within the body. One of these defenses is Anoikis, cell death due to lack of attachment to the extracellular matrix. The development of Anoikis resistant is key in the process of metastasis, because of the need of the cells to detach and circulate the body before settling at a new site within the body. The inhibition of Anoikis resistance development in CRC could lead to improve patient outcome and the failure of metastasis to develop in the lifetime of the disease in the patient. Thus, characterizing and developing methods of inhibition of Anoikis resistance could lead to the decrease in cancer related deaths in the United States as well as finding effect therapeutic options. One of the key characteristics of Anoikis resistance in cells is the develop of stem cell like characteristics, and expression of key stemness factors of Sox2, Oct4, and NANOG. The expression of these factors allows for the cells to overcome their need to be attached to the ECM and travel through the blood stream. We have found through analysis that the membrane bound muicin MUC13 interacts with Sox2 increasing cell stemness leading to the development of anoikis resistance in Colorectal Cancer. Cell cycle analysis of Sw620 cells with high endogenous MUC13 expression showed an 80% less cell death when compare to isogenic Sw480 cells after 48 hours. Western blot anaylsis of the time points 0,24,36, and 48 hrs showed an increase in Sox2 expression and MUC13 expression in Sw620 cells during 24 and 36hrs. To further understand this relationship MUC13 knockdown and overexpressing cells lines will be analyzed to see if any there is a change in the Anoikis phenotype of Sw620 and Sw480 cells. This will also be done with Sox2 knockdown and overexpression. We will also test different therapeutic options, specifically a novel non-viral delivery of MUC13 Crispr Cas9 plasmid to see it effect on Anoikis resistances. Citation Format: Kyle Doxtater, Chidi Zacheaus, Fatemeh Keramatnia, Mehdi Chaib, Manish Triparthi, Subhash Chauhan. MUC13 interacts with SOX2 leading to cell stemness and Anoikis resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2647.

Objective: Colorectal cancer (CRC) is a leading cause of cancer mortality affecting over a million people every year. Biological markers for early detection and distant metastatic disease in patients with CRC are not well defined. We have identified transmembrane mucin MUC13, which is expressed in normal colon mucosa to be highly expressed in colorectal cancer, but the underlying pathways and the signaling mechanisms involved in CRC pathogenesis are not known. Our studies suggest MUC13 correlation with demographic and clinicopathologic characteristics. Materials and Methods: Retrospective institutional tumor registry was reviewed to identify patients with resected colon adenocarcinoma. Archived FFPE tissue blocks were reviewed by an experienced pathologist. Selected representative tissue blocks were serially sectioned at 4 µm. IHC staining was performed using an in-house MAb for MUC13. Slides were digitally scanned and analyzed qualitatively as well as quantitatively using a modified H-score based on the intensity of expression and percentage of stained cells. MUC13 was correlated with disease stage, aggressiveness and inflammatory markers, indicating poor prognosis in CRC patients. MUC13 splice variants were probed using novel designed probes. MUC13 SNPs were identified using unbiased approach by analyzing dpGAP database. Results: 196 tissues, of which 56.1% were female, 52% were white, and the median age at resection was 70. 38 (19.4%) were stage I, 64 (32.7%) stage II, 84 (42.9%) stage III, and 10 (5.1%) stage IV. 100% of colon adenocarcinoma tissues stained positively for MUC13, including definitive tumor epithelial staining, little-to-no background stromal staining, and mild staining of adjacent normal colon mucosa. Typical colon adenocarcinoma cells exhibited strong apical membranous staining with varying degrees of cytoplasmic staining. Advanced stage tumors were noted to more frequently exhibit basolateral and/or circumferential membranous staining compared to early stage tumors which more frequently displayed apical membrane staining alone. Five MUC13 transcript variants were identified by database analysis. Two of the protein coding MUC13 variants (long and short) showed differential expression in aggressive cell lines and patient tissues. Cyclic turnover between the short and long isoforms were observed during Anoikis resistance, cortisol, alcohol and BAP treatments. SNPs have been associated with increased risk of cancer incidence or fatality. Relevant MUC13 SNPs have been identified through systematic analysis of patient databases (dpGAP database). Verification and correlation of identified SNPs with patient disease stage and prognosis will also be presented. Conclusion: This is the first study to correlate MUC13 expression with disease outcome. Alternative transcripts and single nucleotide polymorphisms gave genetic insight of the role of MUC13 in CRC pathogenesis. Citation Format: Manish K. Tripathi, Chidi Zacheaus, Kyle Doxtater, Zachary Stiles, Fatemeh Keramatnia, Nadeem Zafar, Mahul Amin, Meena Jaggi, Subhash Chauhan. MUC13 is a novel molecular signature, for early detection and metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5178.

Triple negative breast cancer (TNBC) is characterized by absence of the estrogen receptor (ER) and progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) amplification. Such cancers are highly aggressive and frequently metastasize to the lung and brain. Unlike other breast cancer subtypes such as, ER+, PR+ and/or HER2+, TNBCs have no specific targeted-therapeutics; therefore, studies should be directed for development of targeted therapies to treat this condition. Recent studies indicate that Wnt/β-catenin signaling is particularly activated in TNBC and is associated with reduced overall survival in all patients. Therefore, pharmacological targeting of Wnt signaling pathway constitutes an ideal approach for treating TNBC and various Wnt inhibitors are currently in use in clinical trials, such as; ICG-001 in metastatic colon cancer. The inhibitory effects of ICG-001 have not been tested in chemoresistant TNBC PDX tumors. Herein, we report for the first time that ICG-001 compound selectively inhibited the growth of several European American (EA) and African American (AA) triple negative breast cancer subtypes MSL and BSL-1 cell lines both in vitro and in vivo models. To further investigate the precise mechanisms of action in the regulation of Wnt/β-catenin signaling by ICG-001, we performed Western blot analysis, apoptosis assays, cell cycle assays and quantitative real-time reverse transcriptase- polymerase chain reactions in human triple negative breast cancer cells. This compound significantly interfered with Wnt/β-catenin signaling, and its inhibition led to downregulation of important downstream targets such as Axin2, HMGA2, PCNA, c-myc and Cyclin D1, which in turn led to inhibition of proliferation, cell cycle progression and metastasis confirming our previous results too. In addition ICG-001 inhibited the invasion and motility of tumor cells and showed inhibition and prevention of visceral metastatic PDX tumors from both chemoresistant EA and AA women. These results indicate that the Wnt inhibitor ICG-001 could constitute a powerful new chemotherapeutic agent against triple negative breast cancer. Citation Format: Iram Fatima, Ikbale El Ayachi, Chidi Zacheaus, Joseph Kerby Gray, Raya Krutilina, Tiffany N. Seagroves, Susan A. Krum, Gustavo A. Miranda-Carboni. WNT inhibitor ICG-001 prevents visceral metastatic triple negative breast cancer in a chemo-resistant patient derived xenograft -PDX-model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1233. doi:10.1158/1538-7445.AM2017-1233